Developing annual vaccine strategies for reducing the severity and prevalence of influenza outbreaks presently requires vigilant monitoring of currently circulating viral strains. This is due to the rapid mutational rate of the protective hemagglutinin (HA) and neuraminidase on the viral surface. Antibodies raised to prior hemagglutinin and neuraminidase sequences will not confer protection from a newly emerged form. Indeed, a single point mutation might render a vaccine inactive toward the new viral mutant form. Alymova, I C, et al, J Virol, 1998; 72:4472-77; Kodihelli, S, et al, J Virol, 1995; 69:4888-97 Moreover, carefully updated and current data on currently circulating strains the choice of which strains to include in the following season's vaccine must be made six months or more in advance.
The primary source of delay from decision to vaccine delivery is due to quantification of protective hemagglutinin (HA) during the production cycle. Currently, HA is quantified by a single radial immunodiffusion (SRID) assay employing standardized antigens and specific sheep antiserum, and quantified by densitometry of active bands separated by gel electrophoresis. Schild G C, et al., Bull World Health Organ, 1975; 52:223-31; Wood, J, et al, Dev Biol Stand, 1977; 39:193-200. The SRID assay is performed by solubilization of the target viral complex and immunodiffusion of the HA through an agarose gel containing antibodies specific for the HA sequence. HA is quantified by precipitation in the region of immunocomplex formation, the size of which is directly proportional to the amount of HA present in the solution. By comparison to a standard curve of known HA concentration produced by the standardized antigens the amount of a specific subtype of HA is determined. Wood, J M, et al, J Biol Stand, 1977; 5:237-247; Williams, M S, Vet Microbiol, 1993; 37:253-26. However, generation of calibrated reagents such as specific antiserum to the target viral antigen requires at least two-three months. Thus, a large portion of the time required to bring a vaccine to market is devoted to the single step of quantifying viral antigen during the production process. Further, the SRID assay is sensitive to matrix ionic strength and pH reducing the assay's overall accuracy. Willkommen, H., et al, Acta Virol, 1983; 27:407-11. This requirement for specific antiserum to the target viral antigen frustrates vaccine development to other rapidly mutating viruses such as HIV and avian influenza.
The World Health Organization constantly monitors the prevalence of individual influenza strains throughout the world in an effort to predict which strains will be most prevalent in the following year. This information is used to determine which influenza strains to include in the next round of vaccine production. However, the time from decision to the vaccine reaching market can be six months or more. During this time a particular strain may have mutated rendering the produced vaccine inactive and reducing the overall effectiveness of the entire influenza vaccine program. Also, in the event that a viral strain mutates to become highly infective producing severe symptoms, a pandemic situation may occur that the current vaccine program will be unable to adequately respond to due to the minimum of several months from identification of the strain until the first vaccine is available.
A primary source of the delay in vaccine production is in obtaining rapid and accurate quantification of immunogenic antigens, and hence viral concentrations in the vaccine under production. The long time from viral identification to market delivery would be devastating in the event of an influenza pandemic. Thus, it is essential that new methods be developed to shorten production time. Recently, quantitative analysis of hemagglutinin antigen by high performance liquid chromatography (HPLC) was reported. Kapteyn, J C, et al, Vaccine, 2006; 24:3137-44; Garcia-Canas, V, et al, Anal Chem, 2007; 79:3164-72. This assay method capitalizes on trypsin cleavage of HA into HA1 and HA2 strands linked by a single disulfide bond. Simple reduction of this bond allows separation of the HA1 strand from the remainder of the viral components by HPLC. The amount of HA1 is determined by measurement of peak area and comparison to that of a standard curve of known quantified HA1 antigen. However, the amount of standard HA1 antigen must be first determined by the traditional SCID assay. Thus, this assay fails to overcome the traditional SCID assay drawbacks of generating standard reagents and specific antibodies. Further, identification and quantification of three different HA subtypes from complex viral production media is not possible.
Other methods for rapidly identifying particular viral strains are presently being developed. Morrissey and colleagues report a matrix-assisted laser desorption/ionization (MALDI) method of generating a fingerprint of particular viral strains. Morrissey, B, et al, J. Virol Meth, 2007; 145:106-14. By identifying particular antigenic regions in HA1 and HA2, these investigators are able to rapidly monitor the prevalence of particular strains. While this method represents a step forward in determining which strains are to be included in the next round of vaccine production, it does not reduce the time required to produce vaccine.
Thus, there exists a need for an improved process of quantifying viral proteins in the vaccine production cycle. There further exists a need for quantifying viral proteins on a time scale that allows vaccine modification to confer protection against a newly emerging strain.